Ansamitocins

ABSTRACT

Improved purification methods for ansamitocins are disclosed.

FIELD OF THE INVENTION

[0001] This invention relates to processes for the preparation ofansamitocins, in particular ansamitocins which can be converted tomaytansinol.

BACKGROUND OF THE INVENTION

[0002] Highly cytotoxic maytansinoid drugs and their therapeutic usehave been described in U.S. Pat. No. 5,208,020. These drugs can beprepared from ansamitocin precursors produced by fermentation ofmicroorganisms such as Actinosynnema.

[0003] Under defined culture conditions, Actinosynnema spp. such asActinosynnema pretiosum produce a number of related ansamitocins. Themajor product is ansamitocin P-3 with an isobutyryl moiety at the C-3position. Other ansamitocins differing only in the C-3 acyl side chainare produced as minor components, P-1 (ethionyl moiety), P-2 (propionylmoiety), P-3′ (butyryl moiety), P-4 (isovaleryl moiety) and P-4′(valeryl moiety). All these compounds may undergo reductive cleavage toproduce a. common product, maytansinol (form P-0). In addition, a numberof other ansamitocins are produced at low levels, which are modified atother sites in the molecule (hydroxylated or n-demethylated). These donot produce the desired P-0 on deacylation.

[0004] Processes for ansamitocin P-3 production from Actinosynemma spp.have been described in U.S. Pat. Nos. 4,162,940; 4,228,239; 4,356,265;and 4,450,234. In general, these methods require adding a filter aid anda water-miscible solvent to whole fermentation broth, removing solidsand extracting the aqueous fraction with a water-immiscible solvent,concentrating and precipitating with petroleum ether, purifying theprecipitate using silica chromatography, and crystallizing followed byfurther chromatography or recrystallization. Alternative processesutilize Diaion HP-10 adsorption instead of silica chromatographyfollowed by further solvent extraction and crystallization.

[0005] These processes can be used to gain acceptable yields ofansamitocin P-3, but the methods involve a large number of stages whichintroduce limitations in large-scale production operations, particularlybecause of the extremely toxic nature of the ansamitocin compounds andthe necessity of ensuring the safety of the human operators of theprocesses. Thus, a need exists to have a safer alternative procedureavailable, utilizing fewer and more contained stages.

SUMMARY OF THE INVENTION

[0006] One aspect of the present invention is a method for preparingpurified ansamitocins comprising the steps of:

[0007] a. culturing an ansamitocin-producing microorganism in a liquidculture medium;

[0008] b. treating the culture medium to facilitate solvent extaction ofansamitocins;

[0009] c. extracting ansamitocins from the culture medium with anaromatic hydrocarbon solvent;

[0010] e. concentrating the extracted ansamitocins; and

[0011] f. purifying the ansamitocins by crystallization.

[0012] Another aspect of the present invention is a method for preparingpurified ansamitocins comprising the steps of:

[0013] a. culturing an ansamitocin-producing microorganism in a liquidculture medium;

[0014] b. extracting ansamitocins from the culture medium with anaromatic hydrocarbon solvent;

[0015] c. concentrating the extracted ansamitocins; and

[0016] d. purifying the ansamitocins by crystallization.

DETAILED DESCRIPTION OF THE INVENTION

[0017] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

[0018] Methods are provided for preparing purified ansamitocins withouta filtration step. The methods comprise the steps of culturing anansamitocin-producing microorganism in a liquid culture medium, treatingthe culture medium to release ansamitocins from the microorganism intothe culture medium thereby facilitating solvent extraction, extractingansamitocins from the treated culture medium with an aromatichydrocarbon solvent, concentrating the extracted ansamitocins, andpurifying the ansamitocins by crystallization. Alternatively, thetreatment step can be omitted.

[0019] The purified ansamitocins may be reduced to maytansinol andinclude ansamitocin P-3, P-1, P-2, P-3′, P-4 and P-4′. The purifiedansamitocins contain only very low levels of undesirable ansamitocinswith modifications at other sites in the molecule. Preferably, theansamitocin-producing microorganism is Actinosynnema spp. Particularlypreferred is Actinosynnema pretiosum ATCC 31565. Also particularlypreferred is Actinosynnema pretiosum ATCC 31281. The microorganisms canbe grown by fermentation culture techniques well-known to those skilledin the art such as those disclosed in U.S. Pat. No. 4,450,234.

[0020] One embodiment of the method of the invention uses treatment ofthe microorganism to aid release of intracellular ansamitocins andrender cell-associated ansamitocins more amenable to solvent extraction.Exemplary treatment methods include sonication, increased pressure orincreased temperature. Preferably, the treatment is a heat treatment.The heat treatment can be conducted at about 60° C. to about 80° C.Preferably, the heat treatment step is conducted at about 75° C. whichkills the microorganism and facilitates solvent extraction ofansamitocins. In an alternative embodiment, no treatment step is usedprior to extraction since approximately 60% of total ansamitocins can befound in the culture medium.

[0021] Ansamitocins can be extracted from culture medium with the use ofaromatic hydrocarbon solvents. The aromatic hydrocarbons have aparticular selectivity for the ansamitocins over other brothconstituents, which ensures only simple processes are requireddownstream to isolate pure product. Preferably, the aromatic hydrocarbonsolvent is toluene or xylene. Particularly preferred is toluene as thisis more amenable to low temperature evaporation. The properties oftoluene and xylene are such that the solvent and aqueous layers arereadily separated under gravity without the need for mechanicalseparation and thus greater containment of the process and increasedoperator safety are achieved. The extraction can be conducted at about20° C. to about 60° C. Preferably, the extraction is conducted at about45° C. The pH of the aqueous solution prior to extraction should be inthe range 3 to 9. Preferably, the pH is near neutrality, i.e., pH 6 to8.

[0022] In general, one equivalent volume of solvent to whole broth (1:1)is preferred for the extraction. Alternative ratio ranges of 2:1 to 1:4can also be used. The solutions are generally mixed slowly with stirringand the mixture stirred until about >80% of the ansamitocins have beenextracted into the organic layer. Preferably the mixture is left tosettle under gravity at a temperature between 15° C. and 50° C. Thepreferred temperature for settling is about 45° C.

[0023] The organic layer is removed, and concentration of the extract byvolume reduction of the solvent may be carried out in vacuo or by othermethods well-known to those skilled in the art. After volume reduction,the concentrated extract can be optionally dissolved in a polar solventsuch as methanol, and clarified using a membrane filter such as PTFE, ora depth filter such as silica or alumina.

[0024] Crystallization is used to purify the desired ansamitocins,particularly P-3, by reducing the levels of unwanted ansamitocins. Apreferred solvent mixture for crystallization is ethyl acetate andheptane. Such crystallization is performed in a conventional manner. Toaid dissolution of the solid for crystallization, a small volume ofmethanol or similar polar solvent may be added prior to addition ofethyl acetate and then treatment with larger volumes of heptane,optionally with stirring and cooling to afford the crystallized product.The product may be recrystallized following the same procedure.

[0025] Alternatively, prior to crystallization, the impure ansamitocinsextracted from the fermentation broth may be purified using silica gel,e.g., by passing a solution of the solvent extract through a bed ofsilica. Solvents used for the chromatography may be toluene andtoluene-methanol mixes. Other solvents known to those skilled in the artcan also be used. The fractions containing ansamitocin P-3 are pooledand concentrated under reduced pressure.

[0026] Methods are also provided for analysis of ansamitocins by HPLC.Quantitation of ansamitocin P-3 and analysis of ansamitocin ratios areachieved by the methods. These methods were employed in the Examples setforth below.

[0027] Quantitation of ansamitocin P-3 in broth and extraction samplescan be determined on a C18 Waters Q Spherisorb S5 ODS2 column, 4.6×250mm, with a 10 mm guard column. UV detection is at 252 nm and 205 nm. Anisocratic mobile phase of 1 ml/min 60% MeCN (0.05% TFA) in water (0.05%TFA) and a 20 μl injection volume are used.

[0028] Analysis of ansamitocin ratios in downstream process samples canbe determined on a C8 Waters Symmetry Shield column, 3.9×150 mm, with noguard column. UV detection is at 252 nm and 205 nm. A gradient mobilephase of 1 ml/min 35-45% MeCN (0.05% TFA) in water (0.05% TFA) over 30minutes with a 10 minute re-equilibration at 40° C. and a 10 μlinjection volume are used.

[0029] Analysis of ansamitocin ratios in chromatography fractions andfinal product can be determined on a C8 Waters Symmetry Shield column,3.9×150 mm, with no guard column with an LC-MS detection system,atmospheric pressure electrospray ionisation, +ve ion mode. 30V conevoltage Mass detection to positively identify the peaks is achieved byfull scan MS (scan from 600-700 amu with quad 1). MS-MS fragmentation todetermine class of ansamitocin is carried out using the same gradientsystem. A gradient mobile phase of 1 ml/min 35-45% MeCN (0.05% TFA) inwater (0.05% TFA) over 30 minutes with a 10 minute re-equilibration at40° C. and a 10 μl injection volume are used.

[0030] Quantitation of ansamitocin P-3 in waste streams and otherlow-level samples requiring high sensitivity can be determined on a C18Waters Spherisorb S5 ODS2 column, 4.6×250 mm, with a 10 mm guard columnand LC-MS-MS atmospheric pressure electrospray ionisation detectionsystem, +ve ion mode, 30V cone voltage. For determination of structuraltype, the molecular ions of the major species were selected and thefragmentation patterns observed were a predominant 547 ion indicatingN-methylated and a 533 indicating N-demethylated. An isocratic mobilephase of 1 ml/min 60% MeCN (0.05% TFA) in water (0.05% TFA) and a 20 μlinjection volume are used.

[0031] The process of the invention can be used to make cell-bindingagent/maytansinoid complexes which are useful as tumor-activatedpro-drugs. Ansamitocins prepared by the process of the invention canundergo reductive cleavage to maytansinol which can be used as describedin U.S. Pat. No. 5,208,020 to produce N-methyl-L-alanine containingmaytansinoid derivatives. These derivatives are then conjugated tocell-binding agents, preferably antibodies, via various linkers such asa disulfide link.

[0032] An exemplary cell-binding agent/maytansinoid complex can beprepared by a process comprising the following steps:

[0033] (1) reducing ansamitocins prepared by the process of theinvention to maytansinol;

[0034] (2) esterifying maytansinol with N-methyl-L-alanine derivativesto form a disulfide-containing maytansinoid ester;

[0035] (3) reducing the disulfide-containing maytansinoid ester preparedby step (2) to a thiol-containing maytansinoid;

[0036] (4) introducing dithiopyridyl groups into a cell-binding agent;and

[0037] (5) linking the thiol-containing maytansinoid produced by step(3) to the dithiopyridyl cell-binding agent of step (4) by a disulfidelink.

[0038] The present invention will now be described with reference to thefollowing specific, non-limiting examples.

EXAMPLE 1 Extraction of Actinosynnema pretiosum Culture Broth andAnsamitocin Purification

[0039] 37L of whole broth containing the producing strain Actinosynnemapretiosum ATCC 31565 (ansamitocin P-3 titer 86.3 mg/L) were heat-treatedin situ at 75° C. for 60 mins to kill the micro-organism and facilitatesolvent extraction of the ansamitocins. 40L of toluene were added andthe mixture warmed to 45° C. Phases were agitated such that a vortex ofupper toluene phase was drawn into the lower broth phase but withoutemulsification or complete homogenisation of the phases. Extraction wascompleted within 16 hours, and separation under gravity within 2 hours.

[0040] 39L of toluene containing 80 mg/L P-3 was recovered by siphon,and evaporated using a 20L rotary evaporator (bath temperature 40-45°C., rate ˜9L/hr). 11.2 g of mobile oil, containing 3.1 g of P-3; 27.6%w/w) was generated after evaporation. The resulting extract wastransferred to a flask by dissolution in toluene and re-evaporation.(Extraction stage yield=97%)

[0041] The extract was taken up in 120 ml toluene and loaded onto asilica column (Kieselgel 60, 125 ml bed volume packed in toluene, 4 cmdiam.×10 cm) in 375 ml toluene (3 bv). The column was washed with 2 bvtoluene and then eluted with 4×2 bv of 2% MeOH in toluene, followed by12×1 bv of 4% MeOH in toluene. The column was eluted at 40 ml/min andproduced tight bands of color. Fractions 7-10 containing ansamitocin P-3were bulked and evaporated to produce 3.2 g of oily solid containing 2.5g of P-3. This material was analyzed by LC-MS and MS-MS.

[0042] At this stage the product contained 85.1% ansamitocin P-3, and atotal of 93.9% of the desired ansamitocins. (Column stage yield=80.6%)

[0043] The product from the silica column was taken up in 200 mL EtOAcwarmed to 40° C. Heptane (200 mL) was added and the solution allowed tocool. The solution was seeded with 1 mg pure P-3 crystals(crystallization also spontaneously occurred at other sites in theflask). After 4 hours at ambient temperature the supernatant wasanalyzed by HPLC and 0.8 g P-3 (30%) was determined to still be insolution. Further heptane was added (150 ml) and the flask left for afurther 3 hours and re-analysed. This indicated that 0.4 g of P-3 (16%)remained in solution. The flask was left overnight at 4° C. Subsequentanalysis indicated that only 70 mg of P-3 (3%) remained in solution. Themother liquors were removed by aspiration, using a sintered filter lineassembly. The white needle crystals were washed with 2×15 mL 1:3EtOAc:heptane. The crystals were dried in situ under vacuum on a rotaryevaporator at 30° C. for 10 hours. 2.5 g of crystals were obtained.(Crystallization yield=86%)

[0044] The final product contained 86% ansamitocin P-3, and a total of98.4% of the desired acylated ansamitocins (P-1, P-2, P-3, P-3′, P-4,P-4′).

EXAMPLE 2 Extraction of Actinosynnema pretiosum Culture Broth andAnsamitocin Purification

[0045] 1,100L of whole broth containing the producing strainActinosynnema pretiosum ATCC 31565. (ansamitocin P-3 titer 75.1 mg/L,82.6 g P-3) were heat-treated in situ at 75° C. for 60 mins to kill themicroorganism and facilitate solvent extraction of the ansamitocins.77.6 g of P-3 remained after the heat kill process. An equal volume oftoluene pre-warmed to 45° C. was added and the mixture was maintained at45° C. Phases were agitated such that a vortex of upper toluene phasewas drawn into the lower broth phase but without emulsification orcomplete homogenization of the phases. Extraction was carried out for 45hours, followed by separation under gravity which occurred within 30min. (Extraction stage yield 90.3%.)

[0046] 1,127L of the toluene extract, containing the ansamitocins, wereconcentrated to 22L using a falling film evaporator (FFE). Theconcentrate was transferred to a 50L rotary evaporator and evaporated tolow volume (evaporation rate 14.6L/hr). The FFE was rinsed with 2×20Ltoluene and the rinsings passed to the evaporator to ensure completetransfer of product. The concentrate was evaporated to dryness.

[0047] The dry extract was taken up in 7.2L of 4% methanol in tolueneand loaded onto a silica column (Kieselgel 60, 4.8L bed volume packed in4% methanol in toluene, 15.0 cm diam.×27.0 cm, loading rate 120 mL/min).The column was eluted isocratically using 4% methanol in toluene at aflow rate of 227-384 mL/min and produced tight bands of color. Initialfractions were one bed volume; fractions 3 to 6 were collected as halfbed volumes. Fractions were monitored by TLC (Kieselgel 60 F₂₅₄ plates,run in 5% methanol in dichloromethane, visualized by UV at 254 nm) andthose containing ansamitocins were monitored by HPLC and LC-MS. Fractionselection for crystallization was based on HPLC and LC-MS analysis ofthe fractions to optimize ansamitocin P-3 recovery and minimizeundesired ansamitocins. Fractions 5 to 10 containing ansamitocin P-3were bulked and evaporated to produce a solid containing 54.6 g of P-3.At this stage the product contained 77.1% ansamitocin P-3, and a totalof 96.1% of the desired ansamitocins. (Column stage yield=92.5%)

[0048] The product from the silica column was taken up in 91 mL methanolpreviously warmed to 46° C., followed by 546 mL ethyl acetate warmed tothe same temperature. Further aliquots of methanol were added to aiddissolution of the solid. A total of 166 mL of methanol was added to themixture. Heptane (100 mL) warmed to 50° C. was added to the first signof cloudiness and then the solution was allowed to cool to ambient ascrystallization commenced. After 4 hours, a further 1,324 mL of heptane(at ambient temperature) was added. The supernatant was analyzed by HPLCand 6.6 g P-3 was determined to still be in solution. The mixture wascooled on ice and further aliquots of heptane (200 and 400 mL) wereadded until 3.9 g of P-3 (6.8%) remained in the mother liquor.

[0049] The mother liquors were removed by aspiration, using a sinteredfilter line assembly. The crystals were washed with 2×150 mL 1:3 ethylacetate:heptane. The crystals were dried in situ on a rotary evaporator,initially under low vacuum, followed by high vacuum (1.0-1.3 mbar) at30° C. for 88.5 hours. 76.4 g of crystals were obtained.

[0050] The final product contained 74.5% ansamitocin P-3 (56.9 g) and atotal of 97.8% of the desired acylated ansamitocins. (Overall yield=69%)

EXAMPLE 3 Silica Chromatography and Crystallization of the Ansamitocins

[0051] 1,008L of Actinosynnema pretiosum whole broth with a titer of64.8 mg/L ansamitocin P-3 were heat treated, extracted with toluene andevaporated essentially as described in Example 1.

[0052] The concentrate containing 34.5 g ansamitocin P-3 was taken up in3 L toluene and loaded onto a silica column (Kieselgel 60, 3.0 L bedvolume, packed in toluene, 13.8 cm diam.×16.6 cm). The column was toppedwith a 4 cm bed of sand. The column was washed with 5L of toluene,followed by 20L of 2% MeOH in toluene, which was collected as 5 Lfractions. The column was then eluted with 20 L of 4% MeOH in toluene,collected as 2.5 L fractions. The ansamitocins were eluted in fractions9 through to 15. Fraction selection for crystallization was based onHPLC and LC-MS analysis of the fractions to optimize ansamitocin P-3recovery and minimize undesired ansamitocins. Fractions 10 to 12 werebulked and evaporated to dryness to yield an oil containing 32.5 g ofansamitocin P-3.

[0053] At this stage the product contained 91.8% ansamitocin P-3, and atotal of 95.3% of the desired acylated ansamitocins. (Column stageyield=94.2%)

[0054] The concentrate from the bulked silica fractions, was warmed in awater bath at 50° C. and dissolved in a minimum volume of warmmethanol/ethyl acetate (50° C.). 60 mL of methanol was added initially,followed by slow addition of 300 mL of ethyl acetate. A further 20 mL ofwarm methanol was added at which point the concentrate was completelydissolved. 200 mL of warm heptane (50° C.) was added and thecrystallization solution removed from the water bath. Crystallizationcommenced, and further heptane was added in 200 mL aliquots until atotal volume of 800 mL had been added. The mixture was cooled in an icebath for 18 hours. The crystallization was monitored by HPLC analysis ofthe mother liquors. At 18 hours 6.3% ansamitocin P-3 remained in themother liquors. A further 200 mL of heptane was added and the mixturecooled for a further five hours. HPLC analysis indicated 4.0%ansamitocins remained in the mother liquors.

[0055] The crystals were recovered by aspiration of the dark brownmother liquors. The crystals were washed three times with 50 ml ofheptane:ethyl acetate, 3:1 and were dried under vacuum (0.8 mBar)overnight. The crystals were uniform, fine, off-white needles containing28.2 g of ansamitocin P-3. (Crystallization stage recovery=86.9%)

[0056] The final product contained 93.1% ansamitocin P-3 and a total of98.4% desired acylated ansamitocins.

EXAMPLE 4 Purification and Crystallization of Ansamitocins from TolueneExtract

[0057] 1,001L of Actinosynnema pretiosum whole broth with a titer of74.5 mg/L ansamitocin P-3 were heat treated, extracted with toluene andevaporated essentially as described in Example 1 to give 9.5 L ofconcentrated extract containing 54.7 g of ansamitocin P-3.

[0058] The concentrate was heated to 45° C. and 14.5 L of heptane wasadded over 33 min to precipitate the ansamitocins. The mixture wascooled on ice. A further 5 L of heptane was added and the mixtureallowed to settle overnight. The mother liquor was removed by aspirationand the precipitate was washed with 10 L of toluene:heptane, 1:1. HPLCanalysis indicated the mother liquor contained 6.5% of ansamitocin P-3,and the wash contained 1.8% P-3. The precipitate was dissolved in 2 L ofmethanol and filtered through a 0.2 micron filter.

[0059] The methanol solution was evaporated to dryness and re-dissolvedin 85 mL of methanol at 50° C. 510 mL of ethyl acetate was added to thesolution followed by 200 mL of heptane. The mixture was cooled toambient and a further 990 mL of heptane was slowly added ascrystallisation commenced. 4.14 g of ansamitocin P-3 remained in themother liquor. A further 400 mL of heptane was added and the mixturecooled on ice. 2.7 g of ansamitocin P-3 remained in the mother liquor.

[0060] The mother liquor was removed by aspiration and the crystals weredissolved in 110 mL of methanol and 520 mL ethyl acetate at 50° C.Heptane was added in two 75 mL aliquots, the mixture was cooled toambient and another 900 mL added. A further 400 mL of heptane was addedand the mixture cooled on ice and left overnight to crystallize. HPLCanalysis indicated 2.3 g of ansamitocin P-3 remained in the motherliquor. The mother liquor was removed by aspiration and the crystalswere washed with heptane:ethyl acetate, 3:1 and were dried under vacuum(0.6 mbar) overnight. The 47.8 g of crystals contained 84.3% ansamitocinP-3, and a total of 97.0% desired acylated ansamitocins.

EXAMPLE 5 Extraction of Actinosynnema pretosium Culture Broth withXylene and Ansamitocin Purification

[0061] 220 mL of heat treated Actinosynnema pretiosum, with a titre of44 ug/ml ansamitocin P-3, were placed in a 45° C. water bath and anequal volume of xylene was added. The phases were mixed such that avortex of xylene was drawn in to the lower broth phase but withoutforming an emulsion. After 20 hours, 64% of the ansamitocin P-3 hadextracted into the xylene phase.

[0062] The mixture was separated under gravity and 170 mL of xylene wereremoved. The xylene extract was evaporated to dryness. The xyleneextract was purified using silica chromatography. The extract wasdissolved in 2 mL of 4% methanol in toluene and loaded and eluted usingthe same solvent mix. Fractions (0.5 mL) were collected and assayed byTLC (Kieselgel 60 F₂₅₄ plates run in dichloromethane:methanol 20:1).Fractions containing ansamitocin P-3 were crystallized using theprocedure described in previous examples. The crystalline product was ofcomparable quality in terms of colour and purity with that produced bytoluene extraction of fermentation broth.

[0063] The present invention may be embodied in other specific formswithout departing from the spirit or essential attributes thereof, and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

1. A method for preparing purified ansamitocins comprising the steps of:a. culturing an ansamitocin-producing microorganism in a liquid culturemedium; b. treating the culture medium to facilitate solvent extractionof ansamitocins; c. extracting ansamitocins from the culture medium withan aromatic hydrocarbon solvent; d. concentrating the extractedansamitocins; and e. purifying the ansamitocins by crystallization.
 2. Amethod for preparing purified ansamitocins comprising the steps of: a.culturing an ansamitocin-producing microorganism in a liquid culturemedium; b. extracting ansamitocins from the culture medium with anaromatic hydrocarbon solvent; c. concentrating the extractedansamitocins; and d. purifying the ansamitocins by crystallization. 3.The method of claim 1 or 2 wherein the ansamitocin producingmicroorganism is Actinosynnema spp.
 4. The method of claim 3 wherein theActinosynnema spp. is Actinosynnema pretiosum ATCC
 31565. 5. The methodof claim 3 wherein the Actinosynnema spp. is Actinosynnema pretiosumATCC
 31281. 6. The method of claim 1 wherein the treatment is heating atabout 75° C.
 7. The method of claim 1 or 2 wherein the solvent istoluene.
 8. The method of claim 7 wherein the ratio of toluene toheat-treated culture medium is about 1:1.
 9. The method of claim 8wherein the extraction is at about 45° C.
 10. The method of claim 1 or 2wherein the solvent is xylene.
 11. The method of claim 10 wherein theratio of xylene to heat-treated culture medium is about 1:1.
 12. Themethod of claim 11 wherein the extraction is at about 45° C.
 13. Themethod of claim 1 or 2 wherein the ansamitocins comprise acylatedansamitocins that can undergo reductive cleavage to form maytansinol.14. Ansamitocins prepared by the process of claim 1 or
 2. 15. Acell-binding agent maytansinoid complex prepared by convertingansamitocins prepared by the process of claim 1 or 2 into thecell-binding agent maytansinoid complex.
 16. The cell-binding agentmaytansinoid complex of claim 15 wherein the cell-binding agent is anantibody.